Instruments called as a cell counting board or a hemocytometer have been used as an easy method for counting of blood cells, cultured cells, or cells isolated with enzymes, or the like. As shown in a perspective illustration of FIG. 11 as an example, this cell counting board comprises a cell counting board body 70 with a similar size of a microscopic glass slide, and a glass plate 80 with a similar size of a cover glass.
The counting board body 70 has two lands 71, 71, and two lands 72, 73 which are located between the land 71 and the land 71. In the central portion of the lands 72, 73, are provided cell counting sections 75, 76 marked off with a grid pattern. The cell counting sections 75, 76 have a role for easy counting of cells as described later. A pitch difference between the lands 71, 71, and the lands 72, 73 is about 0.1 mm; the planes (top faces) of the lands 72, 73 are positioned at a lower position than the planes (top faces) of the lands 71, 71. The glass plate 80 is mounted above the lands 72, 73 so as to attach the glass plate 80 to the lands 71, 71. The glass plate 80 is adhered and fixed to the counting board body 70 (lands 71, 71) usually by a surface tension of water after getting the peripheral portions of the glass plate 80 wet with water, or getting them moisture with expired gas, and putting the glass plate 80 on the counting board body 70, and subsequently pushing the glass plate 80 with fingers strongly.
FIG. 12(a) shows an essential part of grossly enlarged sectional view of the cell counting board 70 attached with the glass plate 80 to the counting board body 70. Because there is a pitch difference between the lands 71, 71, and the lands 72, 73, a space 90 with a constant width (dimension d=0.1 mm) is formed between the plane of the lands 72 (73) and the lower face of the glass plate 80. When the blood diluted or cell suspension is injected into the space 90 with a pipette chip or the like, the liquid fills the space 90 by the surface tension thereof.
On the other hand, the cell counting sections 75, 76 of the cell counting board body 70 are marked off with a grid pattern of thin lines, and the width of the division of the grid pattern is constant. After the cell suspension is injected into the space 90 with a pipette chip or the like, and the liquid fills the space 90 by the surface tension thereof, the cells are observed at the division of the grid pattern, on observation with a microscope or the like. Because the distance of the space at the area of the square part of the division is constant, the rectangular solid with the constant volume is filled with the constant volume of the blood diluted or cell suspension. For example, one assumes the situation where N cells were observed at the area of the square part of the division of 1 mm in width of the cell counting sections 75, 76. In the case of the above space 90 of 0.1 mm in width (d), N cells are present at the rectangular solid with 1 mm×1 mm×0.1 mm. Because 1 mm×1 mm×0.1 mm=0.0001 mL, N cells are present in 0.0001 mL of the injected cell suspension. Therefore, it is easily calculated that the cell suspension injected has the cell density of N×10000/mL.
A definite amount of a blue dye, trypan blue, solution is added to the cell suspension before the injection, the dead cells are stained black-blue dark by intracellular invasion of the blue dye, trypan blue, solution. In contrast, viable cells are not stained black-blue dark, because the viable cells do not allow the intracellular invasion. On this principle, viable cells and dead cells in the cell suspension can be counted separately, by counting the black-blue dark-stained cells and non-stained cells separately.
Conventionally, the material of these cell counting boards has been glass. The cell counting board has been expensive because are needed abrasion works for the purpose of keeping the accurate dimension of the width, and precise works for marking off the accurate lines of the cell counting section 75, 76. Therefore, the cell counting board has been used repeatedly by washing it after use. Expensive glass cell counting boards have been often damaged easily and became unusable by dropping it to the ground. This has been a drawback of the glass cell counting board. In the case where many samples are to be counted for cell numbers, there has been a need for operations where, after counting cells of one sample, the counting board body 70 and the glass plate 80 are washed with water, are washed out the cells and the blue dye, trypan blue, and are wiped off water, and are dried, and subsequently the counting board body 70 and the glass plate 80 are adhered and fixed again. Viable cells die in the blue dye, trypan blue, solution, when they are allowed to stand for 5 minutes. So, the cell counting had to be performed within a few minutes. The above water-washing operations made it difficult to count many samples for a short time. Besides, the operations are troublesome and time-consuming. In the case of research of clinical samples, disposable cell counting boards are in keen demand for the biohazardous handling of infectious bacteria and viruses.
For this purpose, there has been a desire for a disposable cell counting board which is made of plastic resin, and has a space between a counting board body and an upper plate already fixed in advance, and is unnecessary to be assembled by persons dealing with cell counting. A few products for this purpose are known where the counting board body and the upper plate are made of plastic resin and the counting board body and the upper plate are fixed in advance with adhesive (e.g., Patent reference 1).
In the hemocytometer described in this Patent reference 1, as shown in FIG. 12(b), a counting board body 100 and an observation plate 110 are fixed with adhesive 130, and adhesive 130 are mixed with a spacer (spherical particle 131 with a constant particle size) so as to form a clearance 120 with a constant width. This hemocytometer is suitable as a disposable one which can be discarded after use only once.
Patent reference 1: JP-1999-160310-A